How host-like signals drive gene expression and gene expression drives capsule growth in Cryptococcus neoformans [ep_wt]

Cryptococcus neoformans is a fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth in response to host-like conditions. To understand how individual host-like signals affect capsule size and gene expression, we grew cells with and without all combinations of 5 signals suspected of affecting capsule size: Tissue-culture medium (DMEM or RPMI), temperature (37°C), CO2 (5%), and the addition of HEPES buffer. For each combination of conditions, we grew cultures with or without exogenous cAMP, and sampled cells for RNA-Seq at 0, 30, 90, 180, 1440 minutes and for measurement of capsule thickness at 1440 min. We also took samples for RNA-Seq at 30, 90, 180, 1440 minutes and carried out RNA-Seq in quadruplicate. After growth in YPD at 30C, cells were washed in PBS and transferred to one of 48 growth conditions, representing all combinations of 3 media (YPD, DMEM, or RPMI), 2 atmospheres (room air or 5% CO2), 2 temperatures (30C or 37C), +/- 20 mM cAMP, +/- 25 mM HEPES buffer at pH 7.0. Samples were taken for RNA-Seq at 0, 30, 90, 180, 1440 minutes after transfer. We aimed for at least four replicates of each condition at each time point. Replicates were grown and library-prepped on separate days to account for day-to-day variability. On each growth and library prep date, we included cells from between the PBS wash and inoculation into the planned growth conditions, as a constant control and an initial time point. These PBS samples are labeled as time 0. However, they turned out to have much more variability in their RNA-Seq data than other conditions, so we did not use them in our analysis. On 10 dates, we also have controls from just before the PBS wash, which are listed as “medium: YPD, temperature: 30, time: 1440”. We used these as baselines in our time series analyses.